Method for the detection of Salmonella enterica serovar Enteritidis

ABSTRACT

Described herein is the identification of a novel  Salmonella enterica  serovar  Enteritidis  locus that serves as a marker for DNA-based identification of this bacterium. In addition, three primer pairs derived from this locus that may be used in a nucleotide detection method to detect the presence of the bacterium are also disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of United States Provisional Patent Application No. 60/336,089 filed Oct. 31, 2001 and titled “METHOD FOR THE DETECTION OF SALMONELLA ENTERICA SEROVAR ENTERITIDIS THAT IS HIGHLY SPECIFIC.” United States Provisional Patent Application No. 60/336,089 filed Oct. 31, 2001 and titled “METHOD FOR THE DETECTION OF SALMONELLA ENTERICA SEROVAR ENTERITIDIS THAT IS HIGHLY SPECIFIC” is incorporated herein by this reference.

The United States Government has rights in this invention pursuant to Contract No. W-7405-ENG-48 between the United States Department of Energy and the University of California for the operation of Lawrence Livermore National Laboratory.

BACKGROUND

In the last few decades, Salmonella enterica serovar Enteritidis has emerged as a major cause of food-borne illness worldwide. This pathogen is distinguished from its many close relatives also found in poultry environments by its ability to infect chicken ovaries before the eggshell is formed, allowing transmission through intact eggs. Once established in the human host from raw or undercooked eggs or egg products, this bacterium causes gastroenteritis similar to other Salmonella enterica serovars. Infection in poultry flocks, which is asymptomatic, was first noticed in the late 1970's, and in the 1980's spread rapidly throughout the United Kingdom, the United States, South America, and other areas. During this period, the proportion of salmonellosis cases attributed to Salmonella serovar Enteritidis increased substantially, showing a 275-fold increase in Argentina and becoming the predominant cause of this disease in the U.S. (see Hogue, A et al. 1997, Epidemiology and control of Salmonella enteritidis in the United States of America, Revue Scientifique et Technique 16:542-553, Morales, R. A. et al 1999, Economic Consequences of Salmonella enterica Serovar Enteritidis Infection in Humans and the U.S. Egg Industry, Iowa State University Press, Ames, Rodrigue, D. C. et al. 1990, International increase in Salmonella enteritidis: a new pandemic? Epidemiol. Infect. 105:21-27). Baumler et al. suggested that this rapid increase of Salmonella serovar Enteritidis may have been due to successful campaigns to eradicate Salmonella serovars Pullorum and Gallinarum, the causative agents in chickens of bacillary white diarrhea and fowl typhoid, respectively ( see Bäumler, A. J., et al. 2000, Tracing the origins of Salmonella outbreaks, Science, 287:50-2). It is hypothesized that these avian-adapted Salmonellae provided cross-immunity against Salmonella serovar Enteritidis because of important similarities in lipopolysaccharide structures. Therefore, these campaigns may have opened an ecological niche that has since been occupied by Salmonella serovar Enteritidis. This view remains controversial, however, as serovars Gallinarum and Pullorum remain prevalent in many developing countries where serovar Enteritidis has nevertheless increased dramatically, and turkey flocks in developed countries, now free of serovars Gallinarum and Pullorum, have not been colonized by serovar Enteritidis (see Pomeroy, B. S. et al. 1991, Fowl typhoid, In: Calnek, B. W., Barnes, H. J., Beard, C. W. et al. [eds.]; Diseases of Poultry. Ames, IA: Iowa State University Press, pp. 100-7, Silva, E. N. 1985, Salmonella gallinarum problem in Central and South America. In: Snoyenbos, G. H. [ed], and Proceedings of International Symposium on Salmonella, New Orleans, La. American Association of Avian Pathologists, Kennett Square, Pa., pp. 150-6). Unlike the avian-adapted Salmonellae, rodents serve as an animal reservoir for Salmonella serovar Enteritidis, suggesting that culling would not be an effective method of control. It is possible that the use of Salmonella serovar Enteritidis as a rodenticide may have contributed to the current prevalence of this serovar, and it is also likely that infected rodents are currently a source of disease. In addition to the health risks, this pathogen has had a significant economic impact on the egg industry through decreased consumer confidence following well-publicized outbreaks.

SUMMARY OF THE INVENTION

An aspect of the invention includes a method comprising: providing a sample; providing a DNA sequence, wherein the DNA sequence is complimentary to a target DNA sequence derived from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; and detecting the existence of the target DNA sequence by a nucleotide detection method, wherein the existence of the target DNA sequence indicates the presence of salmonella enterica serovar Enteritidis in the sample.

Another aspect of the invention includes a method comprising: providing a sample; providing at least one primer pair derived from a DNA having a sequence of SEQ ID NO: 1; and detecting salmonella enterica serovar Enteritidis with PCR.

A further aspect of the invention includes a method comprising: providing a sample; providing at least one primer pair having a sequence of SEQ ID NO: 5 and SEQ ID NO: 8, SEQ ID NO: 6 and SEQ ID NO: 9 or SEQ ID NO: 7 and SEQ ID NO: 10 and detecting Salmonella enterica serovar enteritidis with PCR.

A further aspect of the invention includes a method comprising: providing a sample; providing an amino acid sequence derived from a DNA sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; and detecting salmonella enterica serovar Enteritidis with PCR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1C shows SEQ ID NO: 1.

FIG. 2 shows SEQ ID NO: 2.

FIG. 3 shows SEQ ID NO: 3.

FIG. 4 shows SEQ ID NO: 4.

FIG. 5 shows SEQ ID NO: 5 and 8.

FIG. 6 shows SEQ ID NO: 6 and 9.

FIG. 7 shows SEQ ID NO: 7 and 10.

FIG. 8 shows an Sdf I amplification product.

FIG. 9 a and 9 b show the amplification of different phage type reference strains using the Sdf1 primer pair in PCR.

FIG. 10 shows a DNA amplification of the Salmonella serovar Enteritidis Sdf I region.

FIG. 11 shows the chromosomal context of Sdf I.

DETAILED DESCRIPTION

Salmonella enterica serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore simple, sensitive and specific detection methods can limit human exposure. Described herein is the identification of a novel Salmonella enterica serovar Enteritidis locus that serves as a marker for DNA-based identification of this bacterium.

Suppression subtractive hybridization (SSH) was used to isolate DNA restriction fragments present in Salmonella serovar Enteritidis, but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis Salmonella enterica isolates comprising 34 different serovars including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one Salmonella difference fragment (Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs can also be used to detect a diverse collection of clinical and environmental Salmonella serovar Enteritidis isolates. In addition, five bacterial genera commonly found with Salmonella serovar Enteritidis were not detected. Treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, showed that Sdf I is located on the chromosome. The Sdf I primers were used to screen a Salmonella serovar Enteritidis genomic library and a unique 4060-bp region was defined. This region was used to develop the detection system for Salmonella serovar Enteritidis described herein. In addition, the 4060-bp region provides sequence information relevant to the unique characteristics of this serovar.

In contrast to other tests, the novel Salmonella enterica serovar Enteritidis locus described herein is not found in a wide range of closely related serovars including Dublin and Pullorum, the two closest relatives of Enteritidis (see Stanley, J. et al. 1994, Phylogenetics of Salmonella enteritidis, International Journal of Food Microbiology. 21:79-87). Thus, the novel Salmonella enterica serovar Enteritidis locus enables highly specific detection of Salmonella serovar Enteritidis. The potential problems associated with plasmid-borne markers are circumvented because the locus has a chromosomal location.

An extensive array of Salmonella serovar Enteritidis phage types from around the world was tested by PCR for the presence of this locus and all phage types associated with human infections were detected. An approximately 7 kb region was isolated by PCR-based screening of a Salmonella serovar Enteritidis library and subsequently sequenced. A primer pair representing the region of this clone not matching the Salmonella enterica serovar Typhi or Paratyphi complete genomes contains six short open-reading frames. The putative proteins show either weak or no similarity to database sequences. Two other primer pairs were developed that are effective at detecting Salmonella serovar Enteritidis. The combined use of these primer pairs can provide tools for developing rapid and specific detection methods for Salmonella enterica serovar Enteritidis.

SUMMARY

The sequence for Salmonella difference region I (Sdr I, SEQ ID NO: 1) from Salmonella enterica serovar Enteritidis CAHFS-285 has been submitted to GENBANK® with accession number AF370716. FIG. 1 shows the sequence of AF370716. Suppression subtractive hybridization was used to find three loci that are restricted to Salmonella enterica serovar Enteritidis or are found in a few close relatives. The three loci are termed Sdf I (SEQ ID NO: 2), Sdf II (SEQ ID NO: 3) and Sdf III (SEQ ID NO: 4). One region, Sdf I, identified as SEQ ID NO: 2 and depicted in FIG. 2, was only found in serovar Enteritidis strains, including a wide range of clinical and environmental samples, and has yielded clear results in laboratory testing. This fact makes this region appropriate for use in the detection of serovar Enteritidis within complex samples. Given the wide range of other Salmonella serovars and other enteric bacteria found in poultry environments, it is desirable to have markers that will distinguish serovar Enteritidis strains from these bacteria. In addition to making an excellent marker for nucleic acid detection, the Sdf I region may also allow the development of an antibody-based test that relies on the detection of one or more putative protein products of the unique ORFs. The extent to which the cloned region varies within serovar Enteritidis strains can confirm that other areas of this region are useful for detection purposes. A phage type 8 strain was tested with three primer pairs spanning the unique region (SEQ ID NO: 2) based on the nucleotide sequence from a phage type 4 strain. For this experiment the three primer pairs were designed to correspond to three locations of Sdf I (i.e. SEQ ID NO: 2), to show that the region is similar in different Enteritidis isolates, and was not just unique to the strain initially examined. The expected products were observed, indicating that the regions complimentary to the primer pairs were also present in phage type 8 strain. An ongoing project at the University of Illinois to sequence the genome of the phage type 8 strain LK5 can allow for a direct sequence comparison of the Sdf I region from two different phage type strains.

Phage typing is currently the standard method for distinguishing subgroups of serovar enteritidis (see Mickman-Brenner, F. W. et al. 1991, Phage typing of Salmonella enteritidis in the United States, J. Olin. Microbiol. 29:2817-23, hereby incorporated by reference). This technique was exploited to ensure that a diverse collection of enteritidis strains was tested with the diagnostic primer pairs disclosed herein. Using the NVSL reference collection, all of 37 phage types were detected with the Sdf I primer pair (SEQ ID NO: 5, and SEQ ID NO: 8 depicted in FIG. 5) except 6A, 9A, 11,16, 20 and 27. Clinical samples for phage types 11, 16, 20 and 27 are not available, indicating that they are not a significant cause of human infections. Although the phage type 6A and 9A reference strains were not detected with the Sdf I primers, two clinical phage type 9A strains and four clinical phage type 6A strains were unambiguously identified by PCR with the Sdf I primer pair. In summary, the Sdf I primer pair clearly detects all strains of a diverse collection of clinical isolates as well as detecting all environmental isolates tested.

A DNA-based test offers the potential for a significant improvement over current methods of Salmonella enterica serovar Enteritidis detection. DNA detection offers the possibility of greater speed, sensitivity and ease. The information disclosed herein can be applied to detection in samples taken directly from poultry environments and comparisons can be made to current detection methods. Combined with improved technology, (e.g., see Belgrader, P., et al. 1999, PCR detection of bacteria in seven minutes, Science, 284:449-50), on-site testing can be performed, thus greatly facilitating detection and regular monitoring for serovar Enteritidis.

The presence of Salmonella enterica serovar enteritidis in a sample can be detected using a nucleotide detection method by providing at least one DNA sequence complimentary to a target DNA sequence derived from SEQ ID NO: 1 (Sdr I shown in FIG. 1A-1C), SEQ ID NO: 2 (Sdf I region shown in FIG. 2), SEQ ID NO: 3 (Sdf II region shown in FIG. 3), or SEQ ID NO: 4 (Sdf III region shown in FIG. 4). The presence of Salmonella enterica serovar enteritidis in a sample can also be detected using polymerase chain reaction (PCR) by providing at least one primer pair derived from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. For example, the primer pairs SEQ ID NO:5 and SEQ ID NO:8 (shown in FIG. 5), SEQ ID NO:6 and SEQ ID NO:9 (shown in FIG. 6) and SEQ ID NO:7 and SEQ ID NO:10 (shown in FIG. 7) are effective. The nucleotide detection method can comprise microarrays, rolling circle amplification (RCA), Southern Blot, transcription mediated amplification (TMA) or flow cytometery.

PCR is a technique utilized to amplify DNA. Typical PCR reactions include appropriate PCR buffers, DNA polymerase and one or more oligonucleotide primers. Various modifications of PCR techniques are possible as detailed in Current Protocols in Molecular Biology ed. F. M. Ausubel, R. Brent, D. D. Moore, K. Struhle, Massachusetts General Hospital and Harvard Medical School (1987) which is hereby incorporated by reference. The following U.S. patents describe PCR and are incorporated herein by reference: U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,800,159. The following U.S. patents describe RCA and are incorporated herein by reference: U.S. Pat. No. 6,344,329; U.S. Pat. No. 6,287,824; U.S. Pat. No. 6,210,884; U.S. Pat. No. 6,183,960; and U.S. Pat. No. 5,854,033. The U.S. Pat. No. 5,399,491 describes TMA and is incorporated herein by reference.

DNA microarray, or DNA chips are fabricated by high-speed robotics, generally on glass but sometimes on nylon substrates, for which probes with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies. An experiment with a single DNA chip can provide researchers information on thousands of genes simultaneously—a dramatic increase in throughput. There are two variants of the DNA microarray technology, in terms of the property of arrayed DNA sequence with known identity: Format I: probe cDNA (500˜5,000 bases long) is immobilized to a solid surface such as glass using robot spotting and exposed to a set of targets either separately or in a mixture. This method, “traditionally” called DNA microarray, is widely considered as developed at Stanford University. A recent article by R. Ekins and F. W. Chu (Microarrays: their origins and applications. Trends in Biotechnology, 1999, 17, 217-218) seems to provide some generally forgotten facts; Format II: an array of oligonucleotide (20˜80-mer oligos) or peptide nucleic acid (PNA) probes is synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The array is exposed to labeled sample DNA, hybridized, and the identity/abundance of complementary sequences is determined. This method, “historically” called DNA chips, was developed at Affimytrix, Inc., which sells its photolithographically fabricated products under the GeneChip® trademark. Many companies are manufacturing oligonucleotide based chips using alternative in-situ synthesis or depositioning technologies. More information on microarrays can be found at the GENE-CHIPS® website.

In one of the embodiment of the invention disclosed herein, Taqman® real time detection may be used for real-time PCR amplification and detection. The principles involved in the conventional Taqman® 5′ exonuclease assay are described in detail by Holland et al in, Detection of specific polymerase chain reaction product by utilizing the 5′—3′ exonuclease activity of Thermus aquaticus DNA polymerase, Proc Natl Acad Sci U S A 88 (16):7276-80, 1991, which is herein incorporated by reference. Taqman® real time detection can also be used to simultaneously detect a plurality of nucleic acid targets when it is used with multiplex PCR, which enables simultaneous detection of more than one target sequence, thus enhancing detection accuracy. Effective target sequences can be derived from DNA sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. A few examples of typical PCR instruments include the ABI PRISM® 7700, the CEPHEID SMART CYCLER®, and the BIO-RAD ICYCLER™.

Isolation of DNA fragments unique to Salmonella enterica serovar Enteritidis. Suppression subtractive hybridization (SSH) was used to identify Salmonella serovar Enteritidis-specific sequences that could serve as diagnostic markers. SSH is a PCR-based technique that enriches for restriction fragments that are present in one strain, termed the tester, but absent in another, termed the driver. Salmonella serovar Enteritidis strain CAHFS-5 was used as the tester (phage type 8), and the closely related serovar Dublin (strain CAHFS-9008117D), also in serogroup D1, was used as the driver. This way, any true SSH products would be likely to distinguish serovar Enteritidis from serovar Dublin and its close relatives. Four restriction enzymes were used in separate SSH experiments: Rsa I, Alu I, Sau3A I and Hae III. Forty-eight clones from each subtraction (192 total) were sequenced, and PCR primers were synthesized for 98 of the products. Ninety-four clones with high similarity to available non-Enteritidis database sequences were not studied further.

PCR amplifications were then performed using the driver and tester DNAs as templates to identify true subtraction products. Nine primer pairs showed amplification with Salmonella serovar Enteritidis but not with serovar Dublin. These unique restriction fragments from which the primers were designed were designated Sdf I to Sdf IX (Salmonella difference fragment). One of the nine fragments was from an SSH experiment using Sau3A I (Sdf I), one was an Alu I fragment, five were Hae III fragments (including Sdf II and Sdf III), and two were Rsa I fragments. The primer pairs (referred to as “Sdf I primer pair” etc.) based on these nine sequences were selected for further analysis.

Characterization of DNA fragments unique to Salmonella serovar Enteritidis. The nine primer pairs that amplified sequences from Salmonella serovar Enteritidis but not serovar Dublin were PCR tested with several other serovars commonly found in the poultry environment to eliminate those primer pairs that were not serovar Enteritidis-specific. Amplification of sequences from one isolate each of Salmonella serovars Typhimurium, Heidelberg, Montevideo, and another isolate of Salmonella serovar Enteritidis (CAHFS-285, a phage type 4 strain) were used for this purpose. In addition, the two strains used in the subtraction (Salmonella serovar Dublin CAHFS-9008117D and Salmonella serovar Enteritidis CAHFS-5) were included as controls. Three of the nine primer pairs detected both strains of serovar Enteritidis but none of the other serovars. These three primer pairs were further evaluated using an extensive collection of Salmonella enterica serovars available at the CAHFS. Eighty-one additional S. enterica isolates were then tested, including 30 additional serovars (for a total of 34 non-Enteritidis serovars including those described above, Table 1) and 12 additional serovar Enteritidis environmental and poultry isolates (Table 2). Most of the 34 non-Enteritidis serovars are encountered at egg production facilities and therefore complicate diagnostic efforts to detect serovar Enteritidis.

The SDf II primer pair (SEQ ID NO: 60 and SEQ ID NO:8 identified seven of the 73 non-Enteritidis isolates, representing sic non-Enteritidis serovars. One of the strains was an isolate of Salmonella serovar dublin, even though this primer pair does not detect the strain of serovar Dublin used for the substraction experiments. This primer pair detected one isolate of Salmonella serovar Worthington, while another isolate of the same serovar was not detected, indicating that there is some degree of diversity within serovars that can be detected by primers form SSH experiments. It is not known if theses differences are due to nucleotide differences in 3′ end of primer-binding site, or whether larger differences are responsible.

Another primer pair, to Sdf III (SEQ ID NO: 7 and SEQ ID NO: 10), amplified a specific product of the predicted size only with the serovar Enteritidis isolates, but amplified other products in six non-Enteritidis isolates (serovars Lomalinda, Mbandaka, Blockley, Derby, Reading and Kentucky) and produced a smear with one isolate (serovar Berta). Clear positive results were obtained with all 14 serovar Enteritidis environmental, poultry and other animal isolates tested in this panel (Table 2). The third primer pair that was tested with this panel of strains was the Sdf I primer pair, which yielded remarkably clear results. No products were amplified from the 73 non-Enteritidis isolates, but all 14 serovar Enteritidis isolates showed a clear band of the expected size.

FIG. 8 shows an Sdf I amplification product for three of the most common phage types of Salmonella serovar Enteritidis (lanes 3-5), while four other Salmonella serovars found in the poultry environment do not show this amplicon (lanes 6-9). Referring to FIG. 8, Lane M, size standards; lane 1, Salmonella serovar Enteritidis CAHFS-546 (phage type 8); lane 2, no template; lane 3, Salmonella serovar Enteritidis CAHFS-184 (phage type 4); lane 4, Salmonella serovar Enteritidis 97-6371A (phage type 8); lane 5, Salmonella serovar Enteritidis 97-1866IN (phage type 13A); lane 6, Salmonella serovar Pullorum; lane 7, Salmonella serovar Typhimurium; lane 8, Salmonella serovar Heidelberg; lane 9, Salmonella serovar Montevideo; lane 10, Escherichia coli; lane 11, Citrobacter freundii. Amplicons produced by the Sdf I primers (293 bp) and the rplI primers (343 bp) are indicated. In addition, two other enteric bacteria, Escherichia coli ATCC 25922 and Citrobacter freundii ATCC 43864 (lanes 10 and 11) are not detected with this primer pair. The Sdf I primer pair was also tested with other bacteria common in poultry environments, namely Proteus mirabilis ATCC 33946, Proteus vulgaris ATCC 13315, Enterobacter aerogenes ATCC 13048, Enterobacter cloacae ATCC 13047, and Providencia rettgeri ATCC 29944, and did not show any amplification.

The Sdf I, Sdf II and Sdf III primer pairs were then used to test 37 NVSL phage type reference strains of Salmonella serovar Enteritidis (Table 3). The Sdf I sequence was present in all but 6 of 37 phage types (PTs 6A, 9A, 11, 16, 20 and 27). No clinical isolates for phage types 11, 16, 20, and 27 are available from the Centers for Disease Control and Prevention (B. Holland, personal communication) suggesting that infections from these phage types are exceedingly rare. A subset of these data is presented in FIG. 9 a. Amplification of 12 different phage types is shown, with only phage type 6A (lane 7) and 9A (lane 10) showing negative results. Referring to FIG. 9 a, Lane M, size markers; lane 1, CAHFS-546 (positive control); lane 2, no template; lane 3, phage type 2; lane 4, phage type 3; lane 5, phage type 4; lane 6, phage type 6; lane 7, phage type 6A; lane 8, phage type 8; lane 9, phage type 9; lane 10, phage type 9A; lane 11, phage type 13A; lane 12, 95-13141 (phage type 14B); lane 13, phage type 24; lane 14, phage type 34. FIG. 9 b shows the detection of Sdf I in phage type reference strains and clinical strains of phage types 6A, 6B and 9A. Lane M, size markers; lane 1, CAHFS-546 (positive control); lane 2, no template; lane 3, NVSL 9 (phage type 6A); lane 4, CAHFS-435 (phage type 6A); lane 5, CAHFS-436 (phage type 6A); lane 6, CAHFS-739 (phage type 6B); lane 7, NVSL 13 (phage type 9A); lane 8, D0144-CDC (phage type 9A); lane 9, D01760-CDC (phage type 9A). Amplicons produced by the Sdf I primers (293 bp) and rplI primers (343 bp) are indicated.

Although these results suggest that the Sdf I primers cannot detect other isolates of phage type 6A or 9A, two clinical isolates of phage type 6A (lanes 4 and 5 of FIG. 9 b) and phage type 9A (FIG. 9 b, lanes 8 and 9) are readily detected with the Sdf I primer pair. Four additional isolates of phage type 6A were also tested, and were detected with the Sdf I primers (Table 2). In addition, one isolate of phage type 6B was also detected (FIG. 9 b, lane 6). These results suggest that strains that are clearly infectious are detected with the Sdf I primers.

The Sdf I and Sdf III primers showed the same pattern when tested with the NVSL strains except for the NVSL phage type 40 reference strain, raising the possibility that the Sdf I and Sdf III difference fragments may be linked in the Salmonella serovar Enteritidis genome. One difference between the Sdf I and Sdf III primer pairs is that the Sdf III primers generate other products for several of the templates that are not the expected size. The Sdf II primer pair showed amplification with all 37 phage types.

The three primer pairs were also tested against 10 additional serovar Enteritidis clinical isolates taken from stool samples of afflicted humans (Table 2). Eight were phage type 4, one was phage type 7, and one was phage type 13. These 10 samples are geographically diverse, having been collected in Spain, Italy, Mexico, and across the United States from Connecticut to Hawaii. All three primer pairs detected the 10 strains.

Combined with the testing of the phage type 6A, 6B and 9A strains discussed above, 16 clinical isolates were tested, and all were detected with the Sdf I primers. Thus, one highly specific marker for Salmonella serovar Enteritidis (Sdf I) has been developed, as well as two other markers that are useful for narrowing Salmonella enterica to just a few serovars.

Database searches with the sequences of Sdf I (333 bp), Sdf II (731 bp) and Sdf III (846 bp) showed that positions 5-274 of Sdf III, when translated, showed high similarity to the deduced amino acid sequence of a hypothetical protein of the putative cryptic phage CP-933R of E. coli O157:H7 strain EDL933 (E value 4×10⁻³⁹). Sdf I and Sdf II showed no similarity to database sequences.

Chromosomal localization of the Sdf I locus. To determine if the Sdf I marker is located on the chromosome or located on a circular plasmid, we developed the following novel assay (FIG. 10). Referring to FIG. 10, the Salmonella serovar Enteritidis Sdf I region is located on the chromosome. Lane 1, total DNA amplified with rplI primers (343-bp amplicon). Lane 2, total DNA amplified with the Sdf I primers (293-bp amplicon). Lane 3, total DNA amplified with the spvC primers (565-bp amplicon). Lane 4, plasmid preparation treated with exo-DNase amplified with rplI primers. Lane 5, plasmid preparation treated with exo-DNase and amplified with Sdf I primers. Lane 6, plasmid preparation treated with exo-DNase and amplified with spvC primers. Lanes 7, 8, 9 are the same as lanes 4, 5, 6, respectively, but without exo-DNase treatment before amplification. Strain CAHFS-285 (phage type 4) was used for these experiments.

A commercially available exodeoxyribonuclease was used to treat plasmid preparations of Salmonella serovar Enteritidis CAHFS-285, a phage type 4 isolate. The enzyme digests contaminating chromosomal DNA present in all plasmid preparations, but does not affect covalently closed or nicked circular DNAs, i.e. circular plasmids. In addition to the Sdf I primer pair, a primer pair to a known chromosomal gene encoding the L9 ribosomal protein (rplI), and a primer pair to a known Salmonella plasmid-borne gene, spvC, were used as controls. Lanes 1-3 show that these primer pairs readily amplify products from total cellular DNA. As expected, all three amplicons were observed in the untreated plasmid preparations (lanes 7-9). In exonuclease treated samples, however, the spvC product (lane 6) showed significant amplification, whereas the rplI (lane 4) and the Sdf I (lane 5) products were only faintly visible. Because the Sdf I signal was reduced similarly to a known chromosomal sequence, this suggests that Sdf I is located on the chromosome.

Cloning of the Sdf I locus. To define the region of the chromosome containing the 333 bp Sdf I SSH product (Salmonella difference region I), a library was constructed using total DNA from the phage type 4 Salmonella serovar Enteritidis strain CAHFS-285. A total of 6,528 E. coli colonies containing plasmids with 4-6 kb inserts, representing greater than 99% of the cellular DNA (assuming a genome size of 5 megabase pairs), were screened by PCR in pools using the Sdf I primer pair. One clone was identified and the complete sequence of its 6,907 bp insert was determined (FIG. 11). Referring to FIG. 11, a schematic representation of the Sdf I region from Salmonella serovar Enteritidis CAHFS-285 (phage type 4). Open boxes indicate sequence with identity to Salmonella enterica serovar Typhi. Gray and black indicate novel sequence. Sdf I, bounded by Sau3A I sites, is shown in black. All open-reading frames greater than 100 codons are indicated with black arrows. There was no similarity to the sequence of Sdf III, which was detected in a similar pattern to that of Sdf I. Sdf I, a Sau3A I fragment isolated by SSH, is found between positions 4928 and 5260 of the genomic clone (black region of FIG. 11). Nucleotide sequence comparisons with database sequences showed a near perfect match at each end to the complete Salmonella enterica serovar Typhi genome. On the left end as shown, the match extends from position 1 to position 2101, and on the right end from position 6160 to 6907. On the left end is a copy of a gene with near-perfect identity to the E. coli ydaO gene. The matches were to two widely separated regions of the serovar Typhi genome (1361375 to 1363475 on the left, 1920934 to 1920189 on the right), suggesting that this region is the site of a major rearrangement with respect to serovar Enteritidis. Overlapping PCR amplifications were used to confirm that the 6,907-bp region of the library clone is contiguous in Salmonella serovar Enteritidis and not the result of the ligation of two or more unrelated fragments (data not shown). There are six open-reading frames greater than 100 codons in the 4060 bp novel region (gray and black bars in FIG. 11). We have designated these six ORFs, lygA-F, for linked to the ydaO gene. These six open reading frames encode possible proteins of 207, 105, 173, 155, 119, and 110 amino acids for lygA-F, respectively. Using a protein BLAST search of the non-redundant database, LygA (position 2161-2784) shows similarity to Exonuclease VIII of Salmonella serovar Typhimurium (E value 2×10⁻⁸). LygC (position 3867-4388) exhibits weak similarity to phage superinfection exclusion protein B of E. coli (E value 6×10⁻⁵), while LygD (position 5036-5503) shows even weaker similarity to phage λ repressor cI (E value 1×10⁻⁴). LygF shows some similarity to a hypothetical protein of prophage CP-933R of E. coli O157:H7, an enterohemorrhagic strain (E value 1×10⁻²²). LygE and F overlap to a large extent, which may indicate that one, the other, or both are not genes. The deduced amino acid sequences of lygB and lygE do not show any similarity to database sequences with a protein BLAST search.

Amplification by PCR was used to examine other areas of the unique region defined by comparison to Salmonella serovar Typhi. Primer pairs to lygA, lygC and lygD were used to amplify sequences from a Salmonella serovar Enteritidis phage type 8 strain (CAHFS-546), and a Salmonella serovar Dublin strain (CAHFS-9008117D), as well as the library strain, Salmonella serovar Enteritidis CAHFS-285 as a positive control. Products of the expected size were observed in the Enteritidis strains, but not the Dublin strain, consistent with the view that the entire unique region is present in Enteritidisstrains, but absent in Dublin strains (data not shown). When primers to the non-unique flanking sequences, which would generate an approximately 4.5 kb amplicon in serovar Enteritidis strains comprising the unique region were used with the serovar Dublin strain mentioned above, an approximately 600 bp product was observed. This may indicate that all or most of the unique region is missing in serovar Dublin, and the locus is otherwise collinear. Sequencing the 600-bp amplicon will help to define the precise nature of the difference between the two serovars.

Materials and Methods

Strains. Strains used are listed in Tables 1, 2 and 3. Some strains, as indicated in the text, were obtained from the American Type Culture Collection (ATCC), Rockville, Md. Serotyping was verified or performed by the California Animal Health and Food Safety Laboratory (CAHFS) using standard procedures. The National Veterinary Services Laboratory (NVSL), Ames, Iowa, performed phage typing by standard methods (see Hickman-Brenner, F. W. et al. 1991, Phage typing of Salmonella enteritidis in the United States. J. Clin. Microbiol. 29:2817-23).

DNA preparation. DNA was isolated from 3 ml cultures after overnight growth in Luria Bertani medium (Sigma, St. Louis, Mo.). Either of two methods was used to purify total DNA, both of which yielded consistent results. DNA STAT-60 isolation reagent (Tel-Test, Friendswood, Tex.) was used according to the manufacturer's recommendations (1 ml per culture). Alternatively, cell pellets were resuspended in 200 μl of TE buffer (10 mM Tris HCl, 1 mM EDTA pH 8.0), and treated with 2.5 μg/ml proteinase K for 30 minutes at 37° C. Successive extractions were performed with saturated phenol, phenol:chloroform, (1:1 v/v) and chloroform:isoamyl alcohol (24:1 v/v). DNA was precipitated with 0.5 ml cold 95% ethanol and 75 μl 3 M sodium acetate pH 5.2, dried under vacuum in a desiccator and resuspended in water.

DNA amplification for strain testing. Oligonucleotide primers (Sigma-Genosys, The Woodlands, Tex.) at 400 nM final concentration were combined with 200 pg genomic DNA template and amplified with Advantage 2 Polymerase (ClonTech, Palo Alto, Calif.). After an initial denaturation at 94° C. for 1 minute, the samples were subjected to 27 cycles of 94° C. for 30s, 58° C. for 30s, and 72° C. for 1 min, followed by a final 7 minute 72° C. incubation. Samples were fractionated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. A primer pair to either the 23S or 16S rRNA gene was used as a positive control for the amplification of each DNA sample, or a primer pair to the rplI gene (encoding the L9 ribosomal protein) was used as an internal control.

Suppression subtraction hybridization, DNA sequencing and analysis. Genome comparisons by suppression subtraction hybridization were performed essentially as described by Akopyants et al. (see Akopyants, N. S. et al. 1998, PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylon, Proc. Natl. Acad. Sci. 95:13108-13, hereby incorporated by reference) with the following exceptions: For subtractions with Sau3A I digested DNA, adaptor 1 was formed by annealing the adaptor 1 long oligonucleotide with the oligonucleotide 5′ GATCACCTGCCCGG (SEO ID NO: 11) to form an adaptor with appropriate cohesive ends. Similarly, adaptor 2 was formed by annealing the adaptor 2 long oligonucleotide with the oligonucleotide 5′ GATCCAATCGGCCG (SEQ ID NO: 12). Ligase (New England Biolabs, Beverly, Mass.) was inactivated by incubation at 72 degrees for 20 minutes. Unpurified PCR products were cloned using the pGEM® T-Easy TA cloning kit (Promega, Madison, Wis.). Recombinant clones were picked using a BioRobotics (Woburn, Mass.) BIOPICK™ automated colony picker, and plasmid templates were prepared by boiling lysis and magnetic bead capture using a high-throughput procedure (see Skowronski, E. W. et al. 2000, Magnetic, microplate-format plasmid isolation protocol for high-yield, sequencing-grade DNA. Biolechniques, 29:786-792, hereby incorporated by reference). Sequencing of plasmid templates was performed using the Applied Biosystems (Foster City, Calif.) Big-Dye™ Terminator system and either ABI PRISM® 377 or 3700 automated sequencers. The sequencing primers used were 5′ TGTAAAACGACGGCCAGT (SEQ ID NO: 13) (forward) and 5′ CAGGAAACAGCTATGACC (SEQ ID NO: 14) (reverse). Sequences were assembled and oligonucleotide primers were designed using the Consed software package (U. Washington, Seattle). Sequence comparisons with the GENBANK® databases were performed using the BLAST (basic local alignment search tool) server at the Baylor College of Medicine (Houston, Tex.) or the server at the National Center for Biotechnology Information (Bethesda, Md.). Both the non-redundant and the unfinished microbial databases were used for comparisons.

Oligonucleotide primers. Sequences of the primer pairs used (Sigma-Genosys, The Woodlands, Tex.) for DNA amplification are as follows: spvC, 5′

spvC, 5′ CTCTGCATTTCACCACCATCACG and (SEQ ID NO: 15) 5′ CTTGCACAACCAAATGCGGAAGAT; (SEQ ID NO: 16) rpll, 5′ GGGTGATCAGGTTAACGTTAAAG and (SEQ ID NO: 17) 5′ CTTCGTGTTCGCCAGTGGTACGC; (SEQ ID NO: 18) 23S, 5′ CTACCTTAGGACCGTTATAGTTAC and (SEQ ID NO: 19) 5′ GAAGGAACTAGGCAAAATGGTGCC; (SEQ ID NO: 20) 16S, 5′ AGAGTTTGATCCTGGCTCAG and (SEQ ID NO: 21) 5′ GGTTACCTTGTTACGACTT; (SEQ ID NO: 22) Sdf I, 5′ TGTGTTTTATCTGATGCAAGAGG and (SEQ ID NO: 23) 5′ CGTTCTTCTGGTACTTACGATGAC; (SEQ ID NO: 24) Sdf II, 5′ GCGAATATCATTCAGGATAAC and (SEQ ID NO: 25) 5′ GCATGTCATACCGTTGTGGA; (SEQ ID NO: 26) Sdf III, 5′ GCTGACTCACACAGGAAATCG and (SEQ ID NO: 27) 5′TCTGATAAGACTGGGTTTCACT. (SEQ ID NO: 28)

Deoxyribonuclease assays. Plasmids were prepared from Salmonella serovar Enteritidis CAHFS-285, by a standard alkaline lysis method (see Birnboim, H. C. et al. 1979, A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7:1513-1523, hereby incorporated by reference) except that proteins and cell debris were precipitated with 7.5 M ammonium acetate (½ volume) instead of sodium acetate. The DNA from a 10 ml culture was resuspended in 40 μl of TE, and 10 μl was digested with PLASMID-SAFE™ DNase (Epicentre Technologies, Madison, Wis.) in a 250 μl reaction with 50 units of enzyme for 5 hours according to the manufacturer's recommendations. Five μl of this reaction were used as a template in PCRs (30 cycles, 1 minute annealing at 65° C., 1 minute extension at 72° C., 30 second denaturation at 94° C.).

Library construction and screening. To construct a genomic library of Salmonella serovar Enteritidis strain CAHFS-285, 100 μg of total DNA was partially digested with 100 units Sau3A I (New England Biolabs, Beverly, Mass.) for 10 minutes. The DNA was fractionated by electrophoresis, and 4-6 kb fragments were excised and gel purified by electroelution. These fragments were ligated to pUC9 (see Vieira, J. et al. 1982, The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers, Gene 19:259-68, hereby incorporated by reference) that had been digested with Barn HI (New England Biolabs, Beverly Mass.), gel purified and treated with shrimp alkaline phosphatase (US Biochemicals, Cleveland, Ohio). Products were introduced into Escherichia coli DH10B cells (Gibco BRL, Rockville, Md.) by electroporation (GENE-PULSER®, BIORAD™, Richmond, Calif.) and transformants were selected with 50 μg/ml ampicillin (Sigma, St. Louis, Mo.) on agar plates with Luria Bertani (LB) medium. Using a BIOPICK™ automated colony picker, white colonies (6,528) were used to inoculate 384-well microfiter plates (Nalge Nunc, Rochester, N.Y.) containing LB with 7.5% (v/v) glycerol, followed by overnight incubation at 37° C. The library was replicated using a 384-pin tool and stored at 70° C. Screening was performed using the Sdf I primers by amplification of combined cultures followed by amplification of single cultures. For each row, 5 μl of each culture was combined and 1 μl of the mixture was PCR tested. For rows with a positive signal, the individual clones were then tested. One clone consistently yielded positive results in PCRs and was selected for sequencing.

DNA sequencing of the Sdf I region. The library clone identified by PCR with the Sdf I primers was purified by alkaline lysis and anion exchange chromatography using a Qiagen (Valencia, Calif.) Plasmid Preparation Kit. The plasmid DNA was digested with Eco RI and Hind III, separated by electrophoresis, and the two insert fragments were gel purified using a Qiaex II kit (Qiagen). The purified fragments were first treated with the Kienow fragment of DNA polymerase I (New England Biolabs) and deoxynucleoside triphosphates, followed by digestion with Alu I, Hae III and Rsa I in separate reactions. Then the products from each of the 3 reactions were separately cloned into pPA9 that had been digested with Eco RV and treated with shrimp alkaline phosphatase. The plasmid pPA9 was constructed by annealing the oligonucleotides 5′ AGCTTGGAATTCGATATCAGGCCTCG (SEO ID NO: 29) and 5′ GATCCGAGGCCTGATATCGAATTCCA (SEO ID NO: 30) which were then cloned between the Hind III and Barn HI sites of pUC9 (see Vieira, J. et al. 1982, The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers, Gene 19:259-68, hereby incorporated by reference). Thirty-two clones from each enzyme sublibrary (96 total) were sequenced as described above and overlapping sequences were assembled with the Consed program to generate the complete sequence of the insert. The assembly was corroborated with restriction mapping based on the sequence.

Nucleotide sequence accession numbers. The sequences for Salmonella difference fragments (Sdf) I-IX from Salmonella enterica serovar Enteritidis CAHFS-5 have been submitted to GENBANK® with accession numbers AF370707-15, respectively. The sequence for Salmonella difference region I (Sdr I) from Salmonella enterica serovar Enteritidis CAHFS-285 has been submitted to GENBANK® with accession number AF370716. FIG. 1A-1C shows the sequence of AF370716. 

1. A method of indicating the presence of Salmonella enterica serovar enteritidis in a sample comprising the steps of: providing the sample, wherein the sample contains or does not contain a target DNA sequence SEQ ID NO: 1, or a target DNA sequence SEQ ID NO: 2, or a target DNA sequence SEQ ID NO: 3, or a target DNA sequence SEQ ID NO: 4; adding primers to the sample, wherein said primers bind to said target DNA sequence SEQ ID NO: 1, or target DNA sequence SEQ ID NO: 2, or target DNA sequence SEQ ID NO: 3, or target DNA sequence SEQ ID NO: 4; amplifying said target DNA sequence SEQ ID NO: 1, or target DNA sequence SEQ ID NO: 2, or target DNA sequence SEQ ID NO: 3, or target DNA sequence SEQ ID NO: 4 that include said bound primers; and detecting the existence of said target DNA sequence SEQ ID NO: 1, or target DNA sequence SEQ ID NO: 2, or target DNA sequence SEQ ID NO: 3, or target DNA sequence SEQ ID NO: 4 by a nucleotide detection method, wherein the existence of said target DNA sequence SEQ ID NO: 1, or target DNA sequence SEQ ID NO: 2, or target DNA sequence SEQ ID NO: 3, or target DNA sequence SEQ ID NO: 4 indicates the presence of Salmonella enterica serovar enteritidis in the sample.
 2. The method recited in claim 1, wherein said nucleotide detection method comprises micro-arrays.
 3. The method recited in claim 1, wherein said nucleotide detection method comprises rolling circle amplification (RCA).
 4. The method recited in claim 1, wherein said nucleotide detection method comprises Southern blot.
 5. The method of indicating the presence of Salmonella enterica serovar enteritidis in a sample recited in claim 1, wherein said step of detecting the existence of the target DNA sequence by a nucleotide detection method comprises Transcription Mediated Amplification (TMA).
 6. The method recited in claim 1, wherein said nucleotide detection method comprises flow cytometery.
 7. The method recited in claim 1, wherein said nucleotide detection method includes a PCR detection method that comprises real-time PCR.
 8. The method recited in claim 7, wherein said primer pair has the sequence of SEQ ID NO: 5 and SEQ ID NO:8 or SEQ ID NO: 6 and SEQ ID NO:9 or SEQ ID NO: 7 and SEQ ID NO:10.
 9. A method of indicating the presence of Salmonella enterica serovar enteritidis in a sample comprising the steps of: providing the sample, wherein the sample contains or does not contain a target DNA sequence SEQ ID NO: 1, or a target DNA sequence SEQ ID NO: 2, or a target DNA sequence SEQ ID NO: 3; adding a primer pair to the sample, wherein said primer pair binds to said target DNA sequence SEQ ID NO: 1, or said target DNA sequence SEQ ID NO: 2, or said target DNA sequence SEQ ID NO: 3, said primer pair has the sequence of SEQ ID NO: 5 and SEQ ID NO:8 or SEQ ID NO: 6 and SEQ ID NO:9 or SEQ ID NO: 7 and SEQ ID NO:10; amplifying said target DNA sequence SEQ ID NO: 1, or said target DNA sequence SEQ ID NO: 2, or said target DNA sequence SEQ ID NO: 3 that include said bound primer pair; and detecting the existence of said target DNA sequence SEQ ID NO: 1, or said target DNA sequence SEQ ID NO: 2, or said target DNA sequence SEQ ID NO: 3, wherein the existence of said target DNA sequence SEQ ID NO: 1, or said target DNA sequence SEQ ID NO: 2, or said target DNA sequence SEQ ID NO: 3 indicates the presence of Salmonella enterica serovar enteritidis in the sample.
 10. The method recited in claim 9, wherein the step of amplifying said target DNA sequence SEQ ID NO: 1, or said target DNA sequence SEQ ID NO: 2, or said target DNA sequence SEQ ID NO: 3 that include said bound primer pair is accomplished using real-time PCR.
 11. A method of indicating the presence of Salmonella enterica serovar enteritidis in a sample comprising the steps of: providing the sample, wherein the sample contains or does not contain a target DNA sequence SEQ ID NO: 1, or a target DNA sequence SEQ ID NO: 2, or a target DNA sequence SEQ ID NO: 3, or a target DNA sequence SEQ ID NO: 4; adding a primer pair to the sample, wherein said primer pair bind to said target DNA sequence SEQ ID NO: 1, or said target DNA sequence SEQ ID NO: 2, or said target DNA sequence SEQ ID NO: 3, or said target DNA sequence SEQ ID NO: 4; said primer pair has the sequence of SEQ ID NO: 5 and SEQ ID NO:8 or SEQ ID NO: 6 and SEQ ID NO:9 or SEQ ID NO: 7 and SEQ ID NO:10; amplifying said target DNA sequence SEQ ID NO: 1, or said target DNA sequence SEQ ID NO: 2, or said target DNA sequence SEQ ID NO: 3, or said target DNA sequence SEQ ID NO: 4; and detecting the existence of said target DNA sequence SEQ ID NO: 1, or said target DNA sequence SEQ ID NO: 2, or said target DNA sequence SEQ ID NO: 3, or said target DNA sequence SEQ ID NO: 4, by a nucleotide detection method, wherein the existence of said target DNA sequence SEQ ID NO: 1, or said target DNA sequence SEQ ID NO: 2, or said target DNA sequence SEQ ID NO: 3, or said target DNA sequence SEQ ID NO: 4 indicates the presence of Salmonella enterica serovar enteritidis in the sample. 